Disposable hand-held device for collection of exhaled breath condensate

ABSTRACT

A breath condensate collection apparatus comprising a central chamber, a breath input assembly, a plunger assembly and a breath condensate collection port. The central chamber has inner and outer side walls with a coolant material sealed in between. The breath input assembly is disposed on the side of the central chamber in fluid communication with the chamber interior. The plunger assembly has a piston, slidably disposed in the chamber, and a handle extending from a first end of the chamber. The collection port is disposed at the second end of the central chamber in fluid communication with the interior of the chamber. Obstructive structures may be arranged in the chamber interior for increasing the surface area on which condensate may form. The apparatus may also include an outlet assembly that may be removed and replaced with a sampling well into which the condensate may be washed with a buffer solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is entitled to the benefit of, and claimspriority to, provisional U.S. Patent Application Serial No. 60/434,916filed Dec. 20, 2002 and entitled “DISPOSABLE HAND-HELD DEVICE FORCOLLECTION OF EXHALED BREATH CONDENSATE FOR ASSAY OF BIOMARKERS FOR THEDETECTION AND PROGNOSIS OF LUNG ISCHEMIA,” and provisional U.S. PatentApplication Serial No. 60/447,581 filed Feb. 14, 2003 and entitled“DEVICE AND METHOD FOR COLLECTION OF EXHALED ALVEOLAR BREATHCONDENSATE,” the entirety of each of which is incorporated herein byreference.

BACKGROUND OF THE PRESENT INVENTION

[0002] 1. Field of the Present Invention

[0003] The present invention relates to the collection of breathcondensate medical testing and diagnosis, and, in particular, to adouble-walled chamber having a coolant material embedded between theinner and outer walls, a side-mounted breath input assembly, an outletfrom which condensate may be collected, and a plunger for expressing thecondensate through the outlet. The collected sample may then be testedfor biomarkers indicating the presence and severity of lung ischemia andassociated pulmonary vasoconstriction.

[0004] 2. Background

[0005] Approximately 6% of exhaled breath is water vapor and waterdroplets. One source of water in breath is from the fluids that line thealveoli of the lung. In other words, the water vapor exhaled from thebreath equilibrates with fluid in the bronchi and alveoli, and thereforebreath condensate collection provides a noninvasive means of samplingthese fluids.

[0006] Exhaled breath condensate contains water soluble and waterinsoluble molecules, including dissolved gases, organic solutes, ionsand proteins. Breath condensate samples from patients with certaindiseases have been shown to contain elevated content of inflammatorymolecules. For example, previous work has demonstrated that smoking,asthma and cystic fibrosis increase the presence of prostaglandinderivatives, thromboxane, leukotrienes and cytokines. (S A Kharitonovand P J Barnes, Exhaled markers of pulmonary disease, Am J Respir CritCare Med 163:1693-1722, 2001.) Until recently, though, little work hasbeen done to identify biomarkers in exhaled breath water vapor that maybe able to assist in determining the presence and severity of lungischemia.

[0007] However, recent research indicates that it may be possible todetect lung ischemia by performing a battery of tests on relativelysmall breath condensate samples. A proposed battery of tests for lungischemia may include fibrinopeptides, thromboxane B2, plateletactivating factor, leukotrienes C, D and E, carbon monoxide-to-nitricoxide ratio and chemokine and other proteins. Measurement offibrinopeptides in breath condensate is believed to have the potentialto allow more localized measurement of the presence of clot in the lungvasulature. It is publicly known that thrombin cleaves fibrinogen Apeptide from fibrinogen as a prerequisite to fibrin gelation. Owing toits small size, it is hypothesized that fibrinopeptides will traversethe alveolar membrane, and equilibrate in alveolar fluid, and thus willbe found in exhaled condensate.

[0008] It is also believed that pulmonary vascular constriction may bedetected by measuring PGF_(2α), thromboxane B₂, PAF, leukotrienes C, D,and E, and the ratio of CO to NO in condensate, thus providing a basisfor initiating pulmonary vasodilator therapy or COX1,2 inhibition. Ourlaboratory has used an experimental pulmonary vascular occlusion (PVO),induced by venous infusion of polystyrene microspheres in a rat, todetermine three major findings related to breath condensate analysis. Weand others have found increased content of PGF_(2α), thromboxane B₂,platelet activating factor (PAF) and vasoconstrictive leukotrienes C, D,and E in the lung washings in our rat model. (Nakos, Am J Resp Crit CareMed 1998, 158:1504) The magnitude of the concentration of thesevasoconstrictive agents correlated with the severity of hypoxemia andpulmonary hypertension. We also have found extremely elevated expressionof the gene encoding heme oxygenase-1 but the nearly complete absence ofexpression of the gene encoding for the inducible enzyme, nitric oxidesynthase. Heme oxygenase produces carbon monoxide (CO) from hemesubstrate whereas nitric oxide synthase produces nitric oxide (NO). Bothare vasodilator substances. In rats subjected to PVO, we have also foundearly increases in lung gene expression of cytokine induced neutrophilattractant 1 and 2 (CINC 1 & 2), and monocyte/macrophage chemoattractantprotein (MCP) 1 and 2, and monocyte/macrophage inflammatory proteins(MIP) 1α and 1β with concomitant increases in each protein in thewashings from the lung airways and alveoli obtained as soon as 2 hoursafter induction of PE, and lasting up to 18 hours after PE induction.The chemoattractant molecules can cause the migration of leukocytes intothe affected area, and through this mechanism, can potentiate injuryduring therapeutic reperfusion.

[0009] Further, the presence of certain chemokines in exhaled condensateis believed to predispose reperfusion injury. The chemokines discoveredin rats included CINC 1, CINC 2, MIP 1α, MIP 1β, and MCP 1 and 2. Thehuman homologues that will be tested in our device will include CXCL1,CXCL 2 and CXCL 3; CCL 2, CCL 3, CCL 4 and CCL 8, using nomenclatureoutlined by Zlotnick and Yoshie, Immunity, 2000, 12:121-127. Chemokineshave been found with an inflammatory model of pulmonary hypertension.(Kimura, Lab Invest 1998 78:571-81; Ikeda, Am J Physiol Heart CircPhysiol, 2002, 283(5):H2021-8). Unlike the in-vivo PVO model, whichcauses primarily obstructed blood flow, the model in the latter studyincites inflammation and remodeling, which over weeks leads to vascularocclusion. Likewise, investigators have also found increased chemokineexpression in lungs subjected to hilar ligation or clamping, whichinterrupts both perfusion and ventilation. The latter model differssignificantly from in-vivo PVO because alveolar ventilation continueswith in-vivo PVO. Thus the ischemic insult differs with in-vivo PVOversus hilar ligation.

[0010] Attempts have been made to analyze exhaled breath, includingbreath condensate, or otherwise measure certain components of exhaledbreath. For example, U.S. Pat. Nos. 6,419,634 and 6,033,368 to Gaston IVet al. disclose a disposable device with a coolant coaxially surroundinga tube in order to cool exhaled breath sufficiently to causecondensation on the walls of the inner tube. Unfortunately, the deviceis designed for the measurement of nitrogen oxides and is not intendedto facilitate protein or eicosinoid determinations on breath condensate.As a result, it suffers from a number of drawbacks. First, the Gastondevice is mounted directly on the analyzer, and thus is too large andtoo cumbersome to use at the bedside for collection of small volumes ofcondensate in emergency department or other ambulatory patients. TheGaston device also suffers from inefficient sample collection inasmuchas the sample must be aggregated in one chamber and then transferred bythe combined actions of droplet accretion and gravity to a separatesecond chamber for analysis. Perhaps worse, the Gaston device isincapable of use separate from the analyzer, in that the apparatuscollects condensate in a chamber specifically designed forspectrophotometric analysis for nitrogen oxides, and thus has no port orother accessible reservoir from which condensate may be aspirated,aliquotted or otherwise withdrawn and subsequently transferred to aseparate assay well to measure the components of the panel describedabove.

[0011] Further, although Gaston mentions the use of a device similar toa syringe plunger in expressing condensed fluid down its inner tube,Gaston fails to solve the problem of how to integrate such a plungerwith the inlet tube. Also, even the inclusion of a plunger to expressfluid down the inner tube of the Gaston device would still fail to solvethe additional problem described previously; that is, Gaston stilldiscloses no simple way to remove the fluid for removing andtransferring the collected fluid for testing outside of the analyzingchamber. Finally, the Gaston coolant is not calibrated to permitcondensation of a calibrated amount of condensate from a limited numberof breaths. Instead, the Gaston device requires a lengthy period ofsustained breathing in order to collect a sufficient quantity ofcondensate, a problem that is exacerbated by the absence of a plunger tomore efficiently remove condensate from the device. This is due in largepart to the considerable quantities of condensate that are necessary inGaston for the intended type of testing to be performed thereon. Asdiscussed previously, a major purpose of the present application is tocollect relatively the small quantities of condensate necessary toperform the types of tests described above. Because such testing was notanticipated by Gaston, the Gaston device was not developed to permitsuch testing. Thus, a need exists for a fast and convenient apparatusand method for collecting small amounts of breath condensate in a mannerthat permits aliquotting as desired for the performance of tests such asthe ones described above.

SUMMARY OF THE PRESENT INVENTION

[0012] It is hypothesized that the ability to measure particularbiomarkers in exhaled breath water vapor, such as the ones describedabove, can assist in determining the presence and severity of lungischemia. Lung ischemia can be caused by multiple processes, includingthromboembolism, sickle cell disease, fat and air embolism. The abilityto collect breath condensate rapidly and easily with a point-of-caredevice would improve the clinical utility of breath-based diagnosis forthis purpose, particularly in the emergency department or clinicsetting. The devices described herein are designed to allow a patient tobreath into a handheld disposable chamber to facilitate the collectionof approximately 100-1000 microliters of aerosolized and vaporized waterand solutes, which can then be analyzed for the presence of specificproteins and other organic compounds, using enzyme-linked immunoassay,and the measurement of the proportion of carbon monoxide relative tonitric oxide using laser spectrophotometry.

[0013] It is an object of the present invention to provide a method ofallowing cold-trapping of exhaled water vapor in a portable device.

[0014] It is another object of the present invention to provide amechanism to permit use of frozen water of a known volume such that theice melts after a known number of exhalations to permit collection ofcondensed exhaled water aerosol and vapor.

[0015] It is yet another object of the present invention to provide abreath condensate collection device having a series of valves to preventcontamination by water vapor and ambient air.

[0016] It is still another object of the present invention to provide abreath condensate collection device using a plunger-type mechanism toexpress collected condensate into a small reservoir to facilitate fluidcollection.

[0017] It is yet another object of the present invention to construct abreath condensate collection device using materials to allow minimalcost of the device such that it is a disposable unit to minimize cost ofthe device.

[0018] It is still another object of the present invention to provide amethod for the collection and aliquotting of a breath condensate samplein an expeditious fashion to facilitate testing for vasoconstrictormolecules, the measurement of the CO-to-NO ratio and the measurement ofchemokine proteins.

[0019] The present invention comprises apparatuses and methods fornon-invasively collecting breath condensate from a patient for testingpurposes. Broadly defined, the present invention according to one aspectis a breath condensate collection apparatus, including: a centralchamber having double side walls and first and second opposing ends,where the double side walls include an inner side wall and an outer sidewall in spaced relationship to one another; a coolant material sealedbetween the inner and outer side walls for cooling at least the innerwalls of the central chamber; a breath input assembly disposed on theoutside of the outer side wall of the central chamber and penetratingboth the inner and outer side walls such that the interior of the breathinput assembly is in fluid communication with the interior of thecentral chamber; a plunger assembly having a piston and a handle, thepiston being slidably disposed in the interior of the central chamber insnug contact with the inner side wall and the handle extending from thefirst end of the central chamber so as to permit the piston to be movedwithin the central chamber; and a breath condensate collection port,disposed at the second end of the central chamber, in fluidcommunication with the interior of the central chamber.

[0020] In features of this aspect, the plunger assembly is adjustablebetween a fully retracted position and a fully depressed position, andwhen the plunger is in its fully retracted position, the fluidconnection between the breath input assembly and the central chamberlies in between the piston and the second end of the central chamber;the location of the breath input assembly on the outside of the outerside wall of the central chamber is adjacent the first end of thecentral chamber; the central chamber includes an end wall at the secondend thereof, and the breath condensate collection port is disposed inthe end wall of the central chamber; the piston includes a surfacefacing toward the second end of the central chamber, a protrusion isdisposed on the surface of the piston, and the protrusion is adapted tofit into the breath condensate collection port when the plunger assemblyis fully depressed into the central chamber; the breath condensatecollection port and the protrusion are each semi-conical in shape; oneor more grooves are disposed in the sides of the protrusion tofacilitate guiding breath condensate toward the breath condensatecollection port; one or more internal passages are disposed in theinterior of the protrusion to facilitate guiding breath condensatetoward the breath condensate collection port; the breath condensatecollection port is disposed on the outside of the outer side wall of thecentral chamber adjacent the second end thereof, the breath condensatecollection port is disposed on the bottom of the central chamber, andthe breath condensate collection apparatus also includes an outlet andan outlet valve disposed on the top of the outer side wall of thecentral chamber adjacent the second end thereof; the breath condensatecollection apparatus also includes a cap for temporarily sealing thebreath condensate collection port; the breath condensate collectionapparatus defines a main axis, the breath input assembly has amouthpiece and a tube structure connecting the mouthpiece to the side ofthe central chamber, and the mouthpiece is generally oriented inparallel to the main axis of the breath condensate collection apparatus;and the breath condensate collection apparatus also includes at leastone clip, mounted at the first end of the central chamber, for lockingthe handle of the plunger assembly in a fully depressed position tofacilitate transport or handling of the breath condensate collectionapparatus until breath condensate collected therein may be removed.

[0021] The present invention, according to another aspect of the presentinvention, is a method of collecting breath condensate, including:providing a central chamber having double side walls, first and secondopposing ends, a coolant material sealed between the inner and outerside walls for cooling at least the inner walls of the central chamber,and a breath condensate collection port disposed at the second end ofthe central chamber; lowering the temperature of the coolant material tochill at least the inner walls of the central chamber; receiving, in theinterior of the central chamber, exhaled breath from a patient,delivered through the inner and outer side walls via a breath inputassembly disposed on the outside of the outer side wall of the centralchamber; condensing portions of the exhaled breath on the inner surfacesof the inner walls of the central chamber; expressing condensate,produced during the condensing step, from the central chamber bydepressing a plunger assembly through the central chamber, therebyforcing the condensate into the breath condensate collection port; andaspirating the expressed condensate from the breath condensatecollection port for analysis thereof.

[0022] In features of this aspect, the aspirating step includesaspirating the expressed condensate into a pipette, and the method alsoincludes transferring the condensate from the pipette to a separateassay well for analysis thereof; the providing step includes providing acentral chamber having an outlet and outlet valve disposed on the top ofthe outer side wall of the central chamber adjacent the second endthereof and having the breath condensate collection port disposed on thebottom of the central chamber, and the method also includes temporarilysealing the breath condensate collection port during the receiving andcondensing steps; and the step of providing includes providing a centralchamber having at least one clip mounted at the first end of the centralchamber, and the method also includes, after expressing the condensateby depressing the plunger assembly, a step of locking, via the at leastone clip, the handle of the plunger assembly in a fully depressedposition to facilitate transport or handling of the breath condensatecollection apparatus until aspirating the expressed condensate.

[0023] The present invention, according to another aspect of the presentinvention, is a breath condensate collection apparatus, including: acentral chamber having double side walls including an inner side walland an outer side wall in spaced relationship to one another; a coolantmaterial sealed between the inner and outer side walls for cooling atleast the inner walls of the central chamber; a breath input assembly,the interior of which is in fluid communication with the interior of thecentral chamber; one or more obstructive structures arranged in theinterior of the central chamber for increasing the surface area on whichcondensate may form; and a breath condensate collection port in fluidcommunication with the interior of the central chamber.

[0024] In features of this aspect, the obstructive structures includegrid-like structures; the obstructive structures include discretegeometric structures; the discrete geometric structures includespherical objects; the discrete geometric structures are formed frommetal; the discrete geometric structures are formed from glass; theobstructive structures are fixed in place within the interior of thecentral chamber; the obstructive structures are free-floating within theinterior of the central chamber; and movement of the obstructivestructures is restricted to a particular portion of the central chamber.

[0025] The present invention, according to another aspect of the presentinvention, is a method of collecting breath condensate, including:providing a double-walled central chamber having one or more obstructivestructures arranged in the interior thereof and a coolant materialsealed between the inner and outer side walls for cooling at least theinner walls of the central chamber; lowering the temperature of thecoolant material to chill at least the inner walls of the centralchamber and the obstructive structures; receiving, in the interior ofthe central chamber, exhaled breath from a patient; condensing portionsof the exhaled breath on the inner surfaces of the inner walls of thecentral chamber and on the obstructive structures; and removingcondensate, produced during the condensing step, from the centralchamber.

[0026] In features of this aspect, the removing step includes washingthe condensate from the obstructive structures; the washing stepincludes adding an amount of liquid to the central chamber, and theamount is selected to correspond to the amount of condensate present inthe central chamber; the washing step includes adding an amount ofliquid to the central chamber, and the amount is selected to correspondto the amount of condensate likely to be present in the central chamberafter the patient exhales into the central chamber for a predeterminedperiod of time; the washing step includes adding an amount of liquid tothe central chamber, and the amount is selected to correspond to theamount of condensate likely to be present in the central chamber afterthe patient completes a predetermined number of exhalations into thecentral chamber; and the method further includes removing theobstructive structures from the central chamber before carrying out thewashing step.

[0027] The present invention, according to another aspect of the presentinvention, is a method of collecting breath condensate, including:providing a double-walled central chamber having a removable outletassembly and a coolant material sealed between the inner and outer sidewalls for cooling at least the inner walls of the central chamber;lowering the temperature of the coolant material to chill at least theinner walls of the central chamber; receiving, in the interior of thecentral chamber, exhaled breath from a patient; condensing portions ofthe exhaled breath on the inner surfaces of the inner walls of thecentral chamber; removing the outlet assembly and replacing it with asampling well; and moving condensate, produced during the condensingstep, from the central chamber to the sampling well.

[0028] In features of this aspect, the step of moving condensateincludes washing condensate from the central chamber into the samplingwell with a known liquid; the step of washing condensate includesintroducing a predetermined quantity of the known liquid into thecentral chamber, mixing the predetermined quantity of the known liquidwith the condensate, and draining the known liquid and the condensatefrom the central chamber into the sampling well; the step of removingthe outlet assembly includes creating an opening into the centralchamber, and the step of introducing includes introducing the knownliquid through the opening; the method further includes storing theknown liquid in the sampling well before replacing the outlet assemblywith the sampling well; and the known liquid is a buffer solution.

[0029] The present invention, according to another aspect of the presentinvention, is a method of collecting breath condensate, including:providing a double-walled central chamber having one or more obstructivestructures arranged in the interior thereof and a coolant materialsealed between the inner and outer side walls for cooling at least theinner walls of the central chamber; calibrating the walls and coolantmaterial of the double-walled central chamber such that, when beginningat a predetermined temperature, a chosen number of breaths, receivedfrom a typical patient, creates a sufficient amount of breath condensateto be collected on the inner surfaces of the inner walls of the centralchamber; lowering the temperature of the coolant material to thepredetermined temperature to chill at least the inner walls of thecentral chamber; receiving, in the interior of the central chamber, aplurality of exhalations from a particular patient, where the number ofexhalations is within a predetermined range established on the basis ofthe chosen number of breaths; condensing portions of the exhalations,received from the patient, on the inner surfaces of the inner walls ofthe central chamber; and removing condensate, produced during thecondensing step, from the central chamber.

[0030] In features of this aspect, the predetermined range is 10 to 25breaths, inclusive; the predetermined temperature is the temperature ofa standard freezer; and the predetermined temperature is 0° F.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Further features, embodiments, and advantages of the presentinvention will become apparent from the following detailed descriptionwith reference to the drawings, wherein:

[0032]FIG. 1 is a side cross-sectional schematic view of a breathcondensate collection apparatus in accordance with a first preferredembodiment of the present invention;

[0033]FIG. 2 is a right end cross-sectional view of the apparatus ofFIG. 1, taken along line 2-2, showing the double-wall construction;

[0034]FIG. 3 is a right end view of the apparatus of FIG. 1;

[0035]FIG. 4 is a side cross-sectional schematic view of the breathcondensate collection apparatus of FIG. 1 with the plunger assembly in afully depressed position;

[0036]FIG. 5 is a side cross-sectional schematic view of a breathcondensate collection apparatus in accordance with a second preferredembodiment of the present invention;

[0037]FIG. 6A is a partial side view of the plunger assembly of FIG. 5,illustrating one type of protrusion;

[0038]FIG. 6B is a right end view of the plunger assembly of FIG. 6A;

[0039]FIG. 7A is a partial side view of the plunger assembly of FIG. 5,illustrating another type of protrusion;

[0040]FIG. 7B is a right end view of the plunger assembly of FIG. 7A;

[0041]FIG. 7C is a partial side cross-sectional view of the plungerassembly of FIG. 7A;

[0042]FIG. 8 is a side cross-sectional schematic view of a breathcondensate collection apparatus in accordance with a third preferredembodiment of the present invention;

[0043]FIG. 9 is a side cross-sectional schematic view of the breathcondensate collection apparatus of FIG. 1, shown in an invertedorientation;

[0044]FIG. 10 is a side cross-sectional schematic view of a breathcondensate collection apparatus in accordance with a fourth preferredembodiment of the present invention;

[0045]FIG. 11 is a cross-sectional schematic view of a removable outletcap for use with the apparatus of FIG. 10;

[0046]FIG. 12 is a cross-sectional schematic view of a removablesampling well for use with the apparatus of FIG. 10; and

[0047]FIG. 13 is a side cross-sectional schematic view of the breathcondensate collection apparatus of FIG. 10 with the sampling wellinstalled thereon.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Referring now to the drawings, in which like numerals representlike components throughout the several views, a variety of breathcondensate collection apparatuses 10, 110, 210, 310, in accordance withthe preferred embodiments of the present invention, is next shown anddescribed.

[0049]FIG. 1 is a side cross-sectional schematic view of a breathcondensate collection apparatus 10 in accordance with a preferredembodiment of the present invention. The breath condensate collectionapparatus 10 includes a double-walled syringe 20 and a breath inputassembly 50. The inner wall 22 of the syringe 20 defines a centralcylinder 24 in which is fitted a plunger assembly 25 that includes apiston 26, a rubber gasket 28 and a handle 30 extending from one end ofthe syringe 20. The outer wall 32 is arranged around the inner wall 22in such a way as to create a narrow space between the inner and outerwalls 22, 32. FIG. 2 is a right end cross-sectional view of theapparatus 10 of FIG. 1, taken along line 2-2, showing the double-wallconstruction, and FIG. 3 is a right end view of the apparatus 10 ofFIG. 1. During manufacture, the space between the inner and outer walls22, 32 may be filled with a jacket of coolant material 34, and the outerwall 32 may then be sealed to the inner wall 22 to prevent leakage. In apreferred embodiment, water may be used as the coolant material 34, butit should be clear that other materials may likewise be used, such aspolyethylene glycol (“PEG”) and the like.

[0050] The syringe 20 further includes an inlet 36, an outlet 38, acollection port 39 and a pair of one-way valves 40, 42. The first valve40 is an intake valve that may be disposed in or adjacent to the inlet36, while the second valve 42 is an exit valve that may be disposed inor adjacent to the outlet 38 in order to facilitate the passage ofexhaled air through the central cylinder 24 in only a single direction.The outlet valve 42 is preferably disposed between the central cylinder24 and the collection port 39 for purposes that will be made evidenthereinbelow. The outlet 38 is preferably disposed at the end oppositethe plunger handle 30 in order to permit materials collected within thecentral cylinder 24 to be expressed through the outlet 38 by the piston26. The valves 40, 42 are illustrated only schematically in the variousdrawings, but they may, for example, include two or three self-sealingleaves formed from plastic or another deformable polymer. The design ofsuch valves would be apparent to those of ordinary skill in the art.

[0051] Further, because the piston 26 fills one end of the syringe 20and the outlet 38 is disposed in the opposite end, the inlet 36 ispreferably arranged to penetrate both the inner and outer walls 22, 32on the side of the syringe 20. In order to cause the most interactionbetween exhaled air passing through the central cylinder 24 and theinner surfaces 44 of the inner wall 22, the inlet 36 is preferablydisposed as close to the piston 26 as possible; however, it will beclear that other arrangements of these components are likewise possiblewithout departing from the scope of the present invention.

[0052] The breath input assembly 50 includes a mouthpiece 52, a filter54 and any tubing 56 necessary to guide exhaled breath from themouthpiece 52 to the inlet 36 of the syringe 20. The mouthpiece 52 is ofsuitable size and shape so as to permit comfortable contact with themouth area of a patient. The filter 54, which may comprise a polymermaterial having perforations or successive intrusions therein, may bearranged within the tubing 56 between the mouthpiece 52 and the syringeinlet 36 to prevent saliva and other liquid or solid matter of a minimumsize from passing therethrough and into the syringe 20. Saliva may befurther prevented from reaching the central cylinder 24 by arranging thebreath input assembly 50 beneath the syringe 20, so that air passingthrough the breath input assembly 50 moves upward. In this arrangement,the effect of gravity on the saliva and other liquid or solid matterhelps to prevent such matter from passing up into the central chamber24, as it instead tends to collect in the tubing 56.

[0053] The tubing 56 is preferably configured so as to avoidinterference between the mouthpiece 52, or any other part of the tubing56, and the operation of the plunger assembly 25, as such operation isdescribed herein. More preferably, the mouthpiece 52 is oriented to begenerally parallel with the syringe 20 and the plunger assembly 25therein, or in other words, the mouthpiece 52 is oriented in parallel tothe main axis defined by the syringe 20. In this orientation, exhaledbreath may be received from a patient without causing interference tothe operation of the plunger assembly 25, and condensate formed on theinside of the syringe 20 as the patient uses the apparatus 10 will tendto drain downward toward the outlet 38.

[0054] The dimensions of the apparatus 10 are chosen so that asufficient volume of condensate may be collected in a relatively shortperiod of time using an apparatus 10 that is small and light enough tobe easily held by a patient or attendant and that does not require thepatient to change his breathing patterns. The walls 22, 32 and otherstructures of the apparatus 10 are preferably constructed of a materialthat tends not to bind to proteins, such as platinum-cured silicon, butother suitable materials may include, but are not limited to, glass,plastic, polyethylene, polycarbonate, or polyvinyl or other syntheticpolymer. The plunger assembly 25 may is likewise preferably constructedfrom a non-protein-binding material, but may be constructed from anysuitable inert material including, but not limited to, plastic, vinyl,polyethylene, rubber, platinum-cured silicon or TEFLON®. In a preferredembodiment, the syringe 20 is between 10 and 20 cm long with a diameterof between 2 and 5 cm, and the collection port 39 is between 5 and 20 mmlong with an internal diameter of between 3 and 10 mm. The thickness ofthe coolant jacket 34 may be between 1 and 10 mm, and the sample volume,expressed from a single use, is preferably between 100 μL and 1000 μL,although it may be possible to obtain useful results from samples assmall as 25 μL.

[0055] In operation, one or more syringes 20 are first stored in arefrigeration device, such as a conventional household or commercialfreezer, that is capable of lowering the temperature to approximately 0°F., thus freezing the jacket of coolant material 34 contained betweenthe inner and outer walls 22, 32 of the syringe 20. When a patient is tobe examined, a single syringe 20 is first withdrawn from the freezer. Ifthe breath input assembly 50 or mouthpiece 52 is stored separately fromthe rest of the apparatus 10, then the apparatus 10 is assembled for useby coupling the various components together. Next, the patient positionsthe mouthpiece 52 in a sealed relationship to his mouth area and exhalesinto the mouthpiece 52. The exhaled breath is guided through the tubing56 and into the central cylinder 24 via the inlet 36. The intake valve40 is forced open by positive pressure, but in the absence of suchpressure it prevents air within the central cylinder 24 from escapingthrough the inlet 36. The exhaled breath then exits through the outlet38 on the end of the cylinder 24 opposite the intake end. The exit valve42 permits air to pass out of the central cylinder 24 only when positivepressure exists on the cylinder side of the valve 42, while in theabsence of such pressure the valve 42 prevents ambient air from enteringthe central cylinder 24 via the outlet 38.

[0056] As the patient exhales through the apparatus 10, the moisture inthe exhaled breath begins to condense on the inner surfaces 44 of thecentral cylinder 24. Because of the depressed temperature of the coolant34 and the syringe 20, the condensate may freeze immediately on theinner surface 44. The collection port 39 is preferably nipple-shaped andapproximately 1 cm long with a diameter small enough to cause someminimal resistance to the exhalation of the patient. The diameter ispreferably chosen so as to slow the rate of expiration such that eachexhalation requires approximately 5 seconds to complete. This increasesthe amount of time for exhaled breath to equilibrate with the insidesurfaces 44 of the central cylinder 24.

[0057] As the patient continues to exhale through the apparatus 10, thefrozen coolant 34 begins to melt. The composition, volume and thicknessof the coolant jacket 34 surrounding the internal cylinder 24 ispreferably calibrated such that the coolant 34 begins to melt afterapproximately 10-15 exhalations by the patient. Once the material 34melts or thaws after the desired number of exhalations, the condensatelikewise can begin to melt. Once the condensate is melted, the plungerassembly 25 may be depressed to express the collected condensate samplethrough the outlet 38 and into the collection port 39. FIG. 4 is a sidecross-sectional schematic view of the breath condensate collectionapparatus 10 of FIG. 1 with the plunger assembly 25 in a fully depressedposition. The location of the exit valve 42 in the outlet 38 between thecentral cylinder 24 and the collection port 39 then permits thecollected condensate to be aspirated from the collection port 39 using aconventional pipette (not shown). The condensate may then be transferredfrom the pipette to a separate assay well for analysis thereof asdesired. Finally, once the condensate has been collected and withdrawnfrom the collection port 39, the entire apparatus 10 may be disposed ofaccording to conventional waste disposition procedures.

[0058] Although not required, the plunger assembly 25 of the breathcondensate collection apparatus 10 may further include one or more clips31 disposed around the end of the syringe 20 opposite the collectionport 39. These clips 31 are preferably formed from a resilient materialand are designed to be deformed away from the syringe 20 as the plungerhandle 30 is forced therebetween. The clips 31 are then biased back intoplace by their natural resiliency once the handle 30 is completelydepressed into the syringe 20 as shown in FIG. 4. Once in this position,the plunger assembly 25 is effectively and conveniently locked in placeby the clips 31, thereby permitting the syringe 20 to be transported andhandled much more safely and reliably. It should be apparent, however,that such clips 31 are not necessary in order to be able to practice thevarious embodiments of the present invention.

[0059]FIG. 5 is a side cross-sectional schematic view of a breathcondensate collection apparatus 110 in accordance with a secondpreferred embodiment of the present invention. This apparatus 110 may beidentical to the apparatus 10 of the first preferred embodiment, exceptthat a semi-conical protrusion 127 (shown schematically in FIG. 5) isdisposed on the surface of the piston 26 facing the outlet 38, thecollection port 139 is sized and shaped to matingly receive theprotrusion 127 therein, and the exit valve 42 is located at the distalend of the collection port 139. The collection port 139 may then beconnected to a colorimetric device designed to detect condensateanalytes.

[0060] Notably, although the exit valve 42 and protrusion 127 are shownonly schematically in the drawings, the exit valve 42 is preferablypositioned as close to the end of the collection port 139 as possible,and the protrusion 127 is preferably sized and shaped so as to abut thevalve 42 when the plunger assembly 25 is fully depressed into thecentral cylinder 24. This design minimizes the amount of fluid that willremain in the syringe 20 when the plunger assembly 25 is fullydepressed, and thus minimizes the amount of condensate that is availablefor subsequent testing.

[0061]FIGS. 6A and 6B are a partial side view and a right end view,respectively, of the plunger assembly 25 of FIG. 5, illustrating onetype of protrusion 127. This type of protrusion 127 includes a series ofgrooves 129 arranged in the surface thereof and extending from the baseof the protrusion 127 to the tip. As the piston 26 is pushed into thevery end of the central cylinder 24, the grooves 129 serve as conduitsfor the condensate remaining in the syringe 20, thus permitting a smallamount of additional condensate to be collected via the collection port139 rather than be trapped in the end of the central cylinder 24. Thiseffect could be further enhanced by the inclusion of additional groovestructures (not shown) in the flat surface of the piston 26 that couldbe used to guide condensate to the grooves 129 in the protrusion 127.

[0062]FIGS. 7A, 7B and 7C are a partial side view, a right end view anda partial side cross-sectional view, respectively, of the plungerassembly 25 of FIG. 5, illustrating another type of protrusion 127. Thistype of protrusion 127 includes a plurality of openings 231 arrangedaround the base of the protrusion 127 and connecting to a hollow shaft233 that exits through the tip of the protrusion 127. The openings 231and central shaft 233 may be between 1 and 2 mm in diameter. Once again,as the piston 26 is pushed into the very end of the central cylinder 24,the openings 231 and hollow shaft 233 serve as conduits for thecondensate remaining in the syringe 20, thus permitting a small amountof additional condensate to be collected via the collection port 139rather than be trapped in the end of the central cylinder 24. Thiseffect could also be further enhanced by the inclusion of additionalgroove structures (not shown) in the flat surface of the piston 26 thatcould be used to guide condensate to the openings 231 in the protrusion127. The central shaft 233 could also be fitted with a rigid but hollowtube structure (not shown) of between 1 and 2 mm (external diameter) toprovide a needle-like extension through which the fluid sample could bedirected and extruded when the plunger assembly 25 is fully depressed.

[0063]FIG. 8 is a side cross-sectional schematic view of a breathcondensate collection apparatus 210 in accordance with a third preferredembodiment of the present invention. Like the apparatuses of the firstand second embodiments 10, 110, this breath condensate collectionapparatus 210 includes a syringe 220, having inner and outer walls 222,232, and a breath input assembly 50. The syringe 220 and the breathinput assembly 50 are generally similar to those of the firstembodiment. However, unlike the first embodiment, the outlet 238 and theoutlet valve 42 are disposed on the top of the syringe 220, near the endopposite the plunger handle 30, and the syringe 220 further includes acollection port 239, disposed on the bottom of the syringe 220 at thevery end of the central cylinder 224, and a cap 37 for covering thecollection port 239. The cap may be threaded or otherwise adapted forsecure connection of the cap 37 to the collection port 239 in order totemporarily close the collection port 239. This apparatus 210 functionsthe same way as the first apparatus embodiment 10 except that exhaledbreath exits through the outlet 238 on the top of the syringe 220, butcondensate may be collected and expressed from the central cylinder 224via the collection port 239 on the bottom of the syringe 220. The cap 37prevents exhaled air from passing through the collection port 239, butmay be unscrewed or otherwise removed when it is desired to collectionthe condensate from the central cylinder 224. This may be particularlyuseful, for example, in expressing condensate directly into an analyzertemporarily or permanently mounted on or adjacent the syringe 220, asdescribed in co-pending and commonly-assigned U.S. Provisional PatentApplication No. 60/447,581, filed Feb. 14, 2003.

[0064] As described previously, it is advantageous for the breath inputassembly 50 in each of the three embodiments 10, 110, 210 discussed thusfar to be arranged beneath the respective syringe 20, 220 in order toaid in preventing saliva and other matter from reaching the centralcylinder or chamber 24, 224. However, it will be apparent that thebreath input assembly 50 may instead be oriented such that the breathinput assembly 50 is located at the top of the syringe 20, 220. FIG. 9is a side cross-sectional schematic view of the breath condensatecollection apparatus 10 of FIG. 1, shown in an inverted orientation,whereby the breath input assembly 50 is disposed at the top of thesyringe 20. Although saliva may be more likely to enter the syringe 20,it should be apparent that other features of the present invention maynot be affected by this arrangement. Although not shown, the apparatuses110, 210 of the second and third embodiments may likewise be rearranged,although it should be noted that the breath input assembly 50 in thethird apparatus 210 would have to be placed on the opposite side of thecollection port 239 in order to facilitate proper operation thereof.

[0065]FIG. 10 is a side cross-sectional schematic view of a breathcondensate collection apparatus 310 in accordance with a fourthpreferred embodiment of the present invention. Like the apparatus 10 ofthe first embodiment, this breath condensate collection apparatus 310includes a double-walled syringe 320 and a breath input assembly 350.The inner wall 322 of the syringe 320 defines a central chamber 324, butunlike the central cylinder 24 of the first embodiment, the centralchamber 324 of the alternative embodiment need not be cylindrical. Theouter wall 332 is arranged around the inner wall 322 in such a way as tocreate a narrow space between the inner and outer walls 322, 332. Likethe apparatus 10 of the first embodiment, however, the space between theinner and outer walls 322, 332 may be filled during manufacture with ajacket of coolant material 34, and the outer wall 332 is then sealed tothe inner wall 322 to prevent leakage.

[0066] The syringe 320 further includes an inlet 36, an outlet cap 60and a first one-way valve 40. The first valve 40 is an intake valve thatmay be disposed in or adjacent to the inlet 36. FIG. 11 is across-sectional schematic view of a removable outlet cap 60 for use withthe apparatus of FIG. 10. The outlet cap 60 includes an outlet 338 and asecond valve 342, which is an exit valve disposed in or adjacent to theoutlet 338 in order to facilitate the passage of exhaled air through thecentral chamber 324 in only a single direction. The outlet cap 60 ispreferably disposed at the end of the central chamber 324 opposite theinlet 36.

[0067] The breath input assembly 350 includes a mouthpiece 52, a filter54 and any tubing 356 necessary to guide exhaled breath from themouthpiece 52 to the inlet 36 of the syringe 320. As with the firstembodiment of the apparatus 10, the mouthpiece 52 is of suitable sizeand shape so as to permit comfortable contact with the mouth area of apatient, and the filter 54 may be arranged within the tubing 356 betweenthe mouthpiece 52 and the syringe inlet 36 to prevent saliva and otherliquid or solid matter of a minimum size from passing therethrough andinto the syringe 320.

[0068] Unlike the embodiments described previously, the apparatus 310 ofthe fourth embodiment does not include a plunger assembly. Instead, asillustrated in FIG. 10, one or more obstructive internal structures 62may be arranged inside the central chamber 324 in order to increase thesurface area with which exhaled breath that passes through the centralchamber 324 may come in contact. Suitable obstructive structures 62 mayinclude grid-like structures and other baffles, spheres such as thoseshown in FIG. 10, or other geometric shapes formed from metal, glass, orother suitable materials. These structures 62 may be held in placewithin the syringe 320 using appropriate screens, bosses or the like(not shown). If cooled in like manner to the syringe 20 of the firstembodiment, breath condensate may subsequently be produced inside thecentral chamber 324 more efficiently.

[0069] However, because the obstructive structures 62 occupy theinterior of the central chamber 324, removal of the condensate collectedthereon may require flushing the interior of the central chamber 324with a suitable buffer solution 72 of known volume and composition. Forexample, the solution may consist of distilled water, or watercontaining an organic dye to indicate the pH of the solution usingvisual or spectrophotometric colorimetry. In a preferred method ofoperation of the alternative embodiment shown in FIG. 10, the apparatus310 is oriented horizontally (not shown), and breath condensate iscollected on the inner surfaces 44 of the central chamber 324 and on theobstructive structures 62 in the same manner as described with respectto the first embodiment. The apparatus 310 may then be rotated to thevertical orientation shown in FIG. 10. With the apparatus 310 in theillustrated orientation, the outlet cap 60 may be removed without dangerof the condensate inside the syringe 320 escaping. Suitable connectionmeans, such as corresponding screw threads or the like, are preferablyprovided on the syringe 320 and the outlet cap 60 to facilitate suchremoval.

[0070] Once the outlet cap 60 has been removed, the buffer solution 72may be added to the central chamber 324 in order to wash the collectedcondensate therefrom. Suitable buffer solutions and volumes will beapparent to those of ordinary skill of the art. FIG. 12 is across-sectional schematic view of a removable sampling well 70 for usewith the apparatus of FIG. 10. With the outlet cap 60 removed and theapparatus oriented as shown in FIG. 10, a sampling well 70 such as thatshown in FIG. 12 may be fastened onto the open end of the syringe 320 toclose that end. FIG. 13 is a side cross-sectional schematic view of thebreath condensate collection apparatus 310 of FIG. 10 with the samplingwell 70 installed thereon. Once the sampling well 70 is in place, theentire apparatus 310 may be inverted to the orientation shown in FIG.13, thus causing the buffer solution 72 to wash the collected breathcondensate out the bottom of the syringe 320 and into the sampling well70. Once collected in the sampling well 70, the sampling well 70 mayonce again be removed and the buffered condensate 74 may be aliquottedfor testing as desired.

[0071] In a variation of the various embodiments described herein, anyof the syringes 10, 110, 210, 310 may alternatively be cooled via anendothermic reaction, such as that created when NH₄NO₃ is hydrated withwater in a 1:4 molar ratio, to produce a temperature below 0° C. forapproximately 10 minutes. This may be facilitated by storing water inthe space between the walls of the syringe 10, 110, 210, 310 inbreakable ampules, surrounded by dry NH₄NO₃, or by sealing only theNH₄NO₃ in the same space, to be hydrated or otherwise injected withwater via a needle port. NH₄NO₃ may be prepared because the reaction maybe triggered by injecting the NH₄NO₃ material with a preset volume oftap water or saline via the needle port, similar to the way a nursewould “flush” an IV line, but it should be apparent that other materialsmay likewise be used to create a suitable endothermic reaction. Forexample, a commercial gel refrigerant that may be activated by slightcompression of the outer wall of the syringe may likewise be used. Onesuch material is Cold Ice, produced by Cold Ice, Inc. of Oakland, Calif.

[0072] Once the sample is obtained using any of the various apparatuses10, 110, 210, 310 described herein, or by some alternative means, abattery of tests may be performed on the sample in order to detect lungischemia. The battery of tests may include measuring or testing forfibrinopeptides, thromboxane B2, platelet activating factor,leukotrienes C, D and E, carbon monoxide-to-nitric oxide ratio andchemokine and other proteins. Each of these has specific purposes, asdescribed below. The various tests may all be performed in a manner wellknown to those of ordinary skill in the art.

[0073] Measurement of fibrinopeptides in breath condensate is believedto have the potential to allow more localized measurement of thepresence of clot in the lung vasulature. As described previously,thrombin cleaves fibrinogen A peptide from fibrinogen as a prerequisiteto fibrin gelation. Owing to its small size, it is hypothesized thatfibrinopeptides will traverse the alveolar membrane, and equilibrate inalveolar fluid, and thus will be found in exhaled condensate. Thus, thepresence of fibrinopeptides in the breath condensate sample wouldindicate the presence of clot.

[0074] Also, pulmonary vascular constriction may be detected bymeasuring PGF₂., thromboxane B₂, PAF, leukotrienes C, D, and E, and theratio of CO:NO in condensate, thus providing a basis for initiatingpulmonary vasodilator therapy or COX1,2 inhibition. Our laboratory hasused an experimental pulmonary vascular occlusion (PVO), induced byvenous infusion of polystyrene microspheres in a rat, to determine threemajor findings related to breath condensate analysis. We and others havefound increased content of PGF_(2α), thromboxane B₂, platelet activatingfactor (PAF) and vasoconstrictive leukotrienes C, D, and E in the lungwashings in our rat model. (Nakos, Am J Resp Crit Care Med 1998,158:1504) The magnitude of the concentration of these vasoconstrictiveagents correlated with the severity of hypoxemia and pulmonaryhypertension. We also have found extremely elevated expression of thegene encoding heme oxygenase-1 but the nearly complete absence ofexpression of the gene encoding for the inducible enzyme, nitric oxidesynthase. Heme oxygenase produces carbon monoxide (CO) from hemesubstrate whereas nitric oxide synthase produces nitric oxide (NO). Bothare vasodilator substances. In rats subjected to PVO, we have also foundearly increases in lung gene expression of cytokine induced neutrophilattractant 1 and 2 (CINC 1 & 2), and monocyte/macrophage chemoattractantprotein (MCP) 1 and 2, and monocyte/macrophage inflammatory proteins(MIP) 1α and 1β with concomitant increases in each protein in thewashings from the lung airways and alveoli obtained as soon as 2 hoursafter induction of PE, and lasting up to 18 hours after PE induction.The chemoattractant molecules can cause the migration of leukocytes intothe affected area, and through this mechanism, can potentiate injuryduring therapeutic reperfision.

[0075] Further, the presence of certain chemokines in exhaled condensateis believed to predispose reperfusion injury. The chemokines discoveredin rats included CINC 1, CINC 2, MIP 1α, MIP 1β, and MCP 1 and 2. Thehuman homologues that will be tested in our device will include CXCL1,CXCL 2 and CXCL 3; CCL 2, CCL 3, CCL 4 and CCL 8, using nomenclatureoutlined by Zlotnick and Yoshie, Immunity, 2000; 12:121-127.Investigators have found chemokines with an inflammatory model ofpulmonary hypertension. (Kimura, Lab Invest 1998-78:571-81; Ikeda, Am JPhysiol Heart Circ Physiol, 2002, 283(5):H2021-8). Unlike the in-vivoPVO model, which causes primarily obstructed blood flow, the model inthe latter study incites inflammation and remodeling, which over weeksleads to vascular occlusion. Likewise, investigators have also foundincreased chemokine expression in lungs subjected to hilar ligation orclamping, which interrupts both perfusion and ventilation. The lattermodel differs significantly from in-vivo PVO because alveolarventilation continues with in-vivo PVO. Thus the ischemic insult differswith in-vivo PVO versus hilar ligation.

[0076] Based on the foregoing information, it is readily understood bythose persons skilled in the art that the present invention issusceptible of broad utility and application. Many embodiments andadaptations of the present invention other than those specificallydescribed herein, as well as many variations, modifications, andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and the foregoing descriptions thereof, withoutdeparting from the substance or scope of the present invention.Accordingly, while the present invention has been described herein indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made merely for the purpose of providing a full andenabling disclosure of the invention. The foregoing disclosure is notintended to be construed to limit the present invention or otherwiseexclude any such other embodiments, adaptations, variations,modifications or equivalent arrangements; the present invention beinglimited only by the claims appended hereto and the equivalents thereof.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for the purpose of limitation.

What is claimed is:
 1. A breath condensate collection apparatuscomprising: a central chamber having double side walls and first andsecond opposing ends, wherein the double side walls include an innerside wall and an outer side wall in spaced relationship to one another;a coolant material sealed between the inner and outer side walls forcooling at least the inner walls of the central chamber; a breath inputassembly disposed on the outside of the outer side wall of the centralchamber and penetrating both the inner and outer side walls such thatthe interior of the breath input assembly is in fluid communication withthe interior of the central chamber; a plunger assembly having a pistonand a handle, wherein the piston is slidably disposed in the interior ofthe central chamber in snug contact with the inner side wall and whereinthe handle extends from the first end of the central chamber so as topermit the piston to be moved within the central chamber; and a breathcondensate collection port, disposed at the second end of the centralchamber, in fluid communication with the interior of the centralchamber.
 2. The breath condensate collection apparatus of claim 1,wherein the plunger assembly is adjustable between a fully retractedposition and a fully depressed position, and wherein when the plunger isin its fully retracted position, the fluid connection between the breathinput assembly and the central chamber lies in between the piston andthe second end of the central chamber.
 3. The breath condensatecollection apparatus of claim 2, wherein the location of the breathinput assembly on the outside of the outer side wall of the centralchamber is adjacent the first end of the central chamber.
 4. The breathcondensate collection apparatus of claim 3, wherein the location of thebreath input assembly is underneath the central chamber.
 5. The breathcondensate collection apparatus of claim 3, wherein the central chamberincludes an end wall at the second end thereof, and wherein the breathcondensate collection port is disposed in the end wall of the centralchamber.
 6. The breath condensate collection apparatus of claim 5,wherein the piston includes a surface facing toward the second end ofthe central chamber, wherein a protrusion is disposed on the surface ofthe piston, and wherein the protrusion is adapted to fit into the breathcondensate collection port when the plunger assembly is fully depressedinto the central chamber.
 7. The breath condensate collection apparatusof claim 6, wherein the breath condensate collection port and theprotrusion are each semi-conical in shape.
 8. The breath condensatecollection apparatus of claim 6, wherein one or more grooves aredisposed in the sides of the protrusion to facilitate guiding breathcondensate toward the breath condensate collection port.
 9. The breathcondensate collection apparatus of claim 6, wherein one or more internalpassages are disposed in the interior of the protrusion to facilitateguiding breath condensate toward the breath condensate collection port.10. The breath condensate collection apparatus of claim 9, furthercomprising a rigid, hollow tube structure extending from one of theinternal passages through which fluid from the interior of theprotrusion may be directed.
 11. The breath condensate collectionapparatus of claim 2, wherein the breath condensate collection port isdisposed on the outside of the outer side wall of the central chamberadjacent the second end thereof.
 12. The breath condensate collectionapparatus of claim 11, wherein the breath condensate collection port isdisposed on the bottom of the central chamber, the breath condensatecollection apparatus further comprising an outlet and an outlet valvedisposed on the top of the outer side wall of the central chamberadjacent the second end thereof.
 13. The breath condensate collectionapparatus of claim 12, further comprising a cap for temporarily sealingthe breath condensate collection port.
 14. The breath condensatecollection apparatus of claim 2, wherein the breath condensatecollection apparatus defines a main axis, wherein the breath inputassembly includes a mouthpiece and a tube structure connecting themouthpiece to the side of the central chamber, and wherein themouthpiece is generally oriented in parallel to the main axis of thebreath condensate collection apparatus.
 15. The breath condensatecollection apparatus of claim 2, further comprising at least one clip,mounted at the first end of the central chamber, for locking the handleof the plunger assembly in a fully depressed position to facilitatetransport or handling of the breath condensate collection apparatusuntil breath condensate collected therein may be removed.
 16. A methodof collecting breath condensate, the method comprising: providing acentral chamber having double side walls, first and second opposingends, a coolant material sealed between the inner and outer side wallsfor cooling at least the inner walls of the central chamber, and abreath condensate collection port disposed at the second end of thecentral chamber; lowering the temperature of the coolant material tochill at least the inner walls of the central chamber; receiving, in theinterior of the central chamber, exhaled breath from a patient,delivered through the inner and outer side walls via a breath inputassembly disposed on the outside of the outer side wall of the centralchamber; condensing portions of the exhaled breath on the inner surfacesof the inner walls of the central chamber; expressing condensate,produced during the condensing step, from the central chamber bydepressing a plunger assembly through the central chamber, therebyforcing the condensate into the breath condensate collection port; andaspirating the expressed condensate from the breath condensatecollection port for analysis thereof.
 17. The method of claim 16,wherein the aspirating step includes aspirating the expressed condensateinto a pipette, the method further comprising: transferring thecondensate from the pipette to a separate assay well for analysisthereof.
 18. The method of claim 17, wherein providing a central chamberincludes providing a central chamber having an outlet and outlet valvedisposed on the top of the outer side wall of the central chamberadjacent the second end thereof and the breath condensate collectionport disposed on the bottom of the central chamber, the method furthercomprising: temporarily sealing the breath condensate collection portduring the receiving and condensing steps.
 19. The method of claim 17,wherein providing a central chamber includes providing a central chamberhaving at least one clip mounted at the first end of the centralchamber, the method further comprising: after expressing the condensateby depressing the plunger assembly, locking, via the at least one clip,the handle of the plunger assembly in a fully depressed position tofacilitate transport or handling of the breath condensate collectionapparatus until aspirating the expressed condensate.
 20. The method ofclaim 17, wherein receiving exhaled breath includes receiving theexhaled breath delivered through the inner and outer side walls via abreath input assembly disposed underneath the central chamber.
 21. Abreath condensate collection apparatus comprising: a central chamberhaving double side walls including an inner side wall and an outer sidewall in spaced relationship to one another; a coolant material sealedbetween the inner and outer side walls for cooling at least the innerwalls of the central chamber; a breath input assembly, the interior ofwhich is in fluid communication with the interior of the centralchamber; one or more obstructive structures arranged in the interior ofthe central chamber for increasing the surface area on which condensatemay form; and a breath condensate collection port in fluid communicationwith the interior of the central chamber.
 22. The breath condensatecollection apparatus of claim 21, wherein the obstructive structuresinclude grid-like structures.
 23. The breath condensate collectionapparatus of claim 21, wherein the obstructive structures includediscrete geometric structures.
 24. The breath condensate collectionapparatus of claim 23, wherein the discrete geometric structures includespherical objects.
 25. The breath condensate collection apparatus ofclaim 23, wherein the discrete geometric structures are formed frommetal.
 26. The breath condensate collection apparatus of claim 23,wherein the discrete geometric structures are formed from glass.
 27. Thebreath condensate collection apparatus of claim 21, wherein theobstructive structures are fixed in place within the interior of thecentral chamber.
 28. The breath condensate collection apparatus of claim21, wherein the obstructive structures are free-floating within theinterior of the central chamber.
 29. The breath condensate collectionapparatus of claim 28, wherein movement of the obstructive structures isrestricted to a particular portion of the central chamber.
 30. Thebreath condensate collection apparatus of claim 21, wherein the locationof the breath input assembly is underneath the central chamber.
 31. Amethod of collecting breath condensate, the method comprising: providinga double-walled central chamber having one or more obstructivestructures arranged in the interior thereof and a coolant materialsealed between the inner and outer side walls for cooling at least theinner walls of the central chamber; lowering the temperature of thecoolant material to chill at least the inner walls of the centralchamber and the obstructive structures; receiving, in the interior ofthe central chamber, exhaled breath from a patient; condensing portionsof the exhaled breath on the inner surfaces of the inner walls of thecentral chamber and on the obstructive structures; and removingcondensate, produced during the condensing step, from the centralchamber.
 32. The method of claim 31, wherein the removing step includeswashing the condensate from the obstructive structures.
 33. The methodof claim 32, wherein the washing step includes adding an amount ofliquid to the central chamber, wherein the amount is selected tocorrespond to the amount of condensate present in the central chamber.34. The method of claim 32, wherein the washing step includes adding anamount of liquid to the central chamber, wherein the amount is selectedto correspond to the amount of condensate likely to be present in thecentral chamber after the patient exhales into the central chamber for apredetermined period of time.
 35. The method of claim 32, wherein thewashing step includes adding an amount of liquid to the central chamber,wherein the amount is selected to correspond to the amount of condensatelikely to be present in the central chamber after the patient completesa predetermined number of exhalations into the central chamber.
 36. Themethod of claim 32, further comprising the step of removing theobstructive structures from the central chamber before carrying out thewashing step.
 37. A method of collecting breath condensate, the methodcomprising: providing a double-walled central chamber having a removableoutlet assembly and a coolant material sealed between the inner andouter side walls for cooling at least the inner walls of the centralchamber; lowering the temperature of the coolant material to chill atleast the inner walls of the central chamber; receiving, in the interiorof the central chamber, exhaled breath from a patient; condensingportions of the exhaled breath on the inner surfaces of the inner wallsof the central chamber; removing the outlet assembly and replacing itwith a sampling well; and moving condensate, produced during thecondensing step, from the central chamber to the sampling well.
 38. Themethod of claim 37, wherein the step of moving condensate includeswashing condensate from the central chamber into the sampling well witha known liquid.
 39. The method of claim 38, wherein the step of washingcondensate includes: introducing a predetermined quantity of the knownliquid into the central chamber; mixing the predetermined quantity ofthe known liquid with the condensate; and draining the known liquid andthe condensate from the central chamber into the sampling well.
 40. Themethod of claim 39, wherein the step of removing the outlet assemblyincludes creating an opening into the central chamber, and wherein thestep of introducing includes introducing the known liquid through theopening.
 41. The method of claim 39, further comprising: beforereplacing the outlet assembly with the sampling well, storing the knownliquid in the sampling well.
 42. The method of claim 38, wherein theknown liquid is a buffer solution.
 43. The method of claim 42, whereinthe buffer solution is distilled water.
 44. The method of claim 42,wherein the buffer solution includes an organic dye to indicate the pHof the solution.
 45. The method of claim 38, further comprising: beforereplacing the outlet assembly with the sampling well, storing the knownliquid in the sampling well.
 46. The method of claim 38, furthercomprising: before replacing the outlet assembly with the sampling well,storing the known liquid in the sampling well.
 47. A method ofcollecting breath condensate, the method comprising: providing adouble-walled central chamber having one or more obstructive structuresarranged in the interior thereof and a coolant material sealed betweenthe inner and outer side walls for cooling at least the inner walls ofthe central chamber; calibrating the walls and coolant material of thedouble-walled central chamber such that, when beginning at apredetermined temperature, a chosen number of breaths, received from atypical patient, creates a sufficient amount of breath condensate to becollected on the inner surfaces of the inner walls of the centralchamber; lowering the temperature of the coolant material to thepredetermined temperature to chill at least the inner walls of thecentral chamber; receiving, in the interior of the central chamber, aplurality of exhalations from a particular patient, wherein the numberof exhalations is within a predetermined range established on the basisof the chosen number of breaths; condensing portions of the exhalations,received from the patient, on the inner surfaces of the inner walls ofthe central chamber; and removing condensate, produced during thecondensing step, from the central chamber.
 48. The method of claim 47,wherein the predetermined range is 10 to 25 breaths, inclusive.
 49. Themethod of claim 47, wherein the predetermined temperature is thetemperature of a standard freezer.
 50. The method of claim 49, whereinthe predetermined temperature is 0° F.